Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology - Part D

journal homepage: www.elsevier.com/locate/cbpd

This article is a part of the Special Issue on Aquaculture Global metabolic responses of the lenok (Brachymystax lenok) to thermal ☆ stress T

Yang Liua, Jiashou Liuc, Shaowen Yec, Dominique P. Bureaud, Hongbai Liua, Jiasheng Yina, ⁎ Zhenbo Moue, Hong Linb, Fuhua Haob, a Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China b Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, China c Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China d Fish Nutrition Research Laboratory, Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada e Institute of Fisheries Science, Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa 850002, China

ARTICLE INFO ABSTRACT

Keywords: High temperature is a powerful stressor for fish living in natural and artificial environments, especially for cold Thermal stress water species. Understanding the impact of thermal stress on physiological processes of fish is crucial for better Metabonomics cultivation and fisheries management. However, the metabolic mechanism of cold water fish to thermal stress is NMR still not completely clear. In this study, a NMR-based metabonomic strategy in combination with high- Multivariate analysis throughput RNA-Seq was employed to investigate global metabolic changes of plasma and liver in a typical cold Brachymystax lenok water fish species lenok (Brachymystax lenok) subjected to a sub-lethal high temperature. Our results showed that thermal stress caused multiple dynamic metabolic alterations of the lenok with prolonged stress, including re- pression of energy metabolism, shifts in lipid metabolism, alterations in amino acid metabolism, changes in choline and nucleotide metabolisms. Specifically, thermal stress induced an activation of glutamate metabolism, indicating that glutamate could be an important biomarker associated with thermal stress. Evidence from Hsp 70 gene expression, blood biochemistry and histology confirmed that high temperature exposure had negative effects on health of the lenok. These findings imply that thermal stress has a severe adverse effect on fish health and demonstrate that the integrated analyses combining NMR-based metabonomics and transcriptome strategy is a powerful approach to enhance our understanding of metabolic mechanisms of fish to thermal stress.

1. Introduction between the optimal range and sublethal threshold forced fish to re- spond to processes that help minimize thermal damage by diverting Fish are poikilotherms that often inhabit thermally variable en- some changes in gene expression, protein abundance and metabolic vironments and many aspects of their biology are acutely attuned to process (Mahanty et al., 2016). water temperature (Logan and Buckley, 2015). At low temperature, Recently, systems biology approaches have been employed to fur- feed intake, growth and metabolism are suppressed, whereas elevated ther elucidate biological processes of fish to thermal stress (Kullgren temperatures correlate with an increase in growth up to an optimal et al., 2013; Jeffries et al., 2014). Previous studies have been carried out point above which thermal stress occurs (Fang et al., 2010). Some to investigate transcriptomic responses to high temperature exposure in studies have shown that thermal stress can negatively affect growth, fish (Lewis et al., 2010; Olsvik et al., 2013). Liver transcriptional data of survival and metabolic processes of fish (Viant et al., 2003; Marine and Atlantic salmon (Salmo salar) exposed to chronic high temperature Cech Jr, 2004; McCullough et al., 2009). For salmonid species, the showed that thermal stress significantly down-regulated some tran- optimal water temperature range for optimal feeding and growth is scripts encoding proteins involved in the protection against oxidative generally below 20 °C, and mortality can occur when the temperature stress and some other stress markers such as HIF1A, CYP1A, MTOR and exceed 25 °C (Wang et al., 2016). However, exposure to temperatures PSMC2 (Olsvik et al., 2013). In contrast to a cooler temperature, a high

☆ This article is part of a special issue entitled: Aquaculture- edited by Dr. Matt Rise, Dr. Muyan Chen and Dr. Chris Martyniuk. ⁎ Corresponding author. E-mail address: [email protected] (F. Hao). https://doi.org/10.1016/j.cbd.2019.01.006 Received 11 October 2018; Received in revised form 17 December 2018; Accepted 9 January 2019 Available online 15 January 2019 1744-117X/ © 2019 Elsevier Inc. All rights reserved. Y. Liu et al. Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319 temperature of 19 °C caused two Pacific salmon species to reveal dif- employed to assess the changes of gene expressions and validate the ferential gene expressions, such as protein folding, protein synthesis, metabolic alterations associated with thermal stress. This integrated metabolism, oxidative stress and ion transport (Jeffries et al., 2014). At study was conducted to investigate global metabolic responses of the an acute heat stress (1 h at 25 °C followed by recovery at 11 °C), the lenok to sub-lethal high temperature by combining NMR-based meta- transcripts of the rainbow trout (Oncorhynchus mykiss) displayed bonomics, transcriptomics, and some conventional analyses. Our results changes in genes involved in the stress response (Hsp group), immune are useful to gain novel insights into the habitual nature of the lenok to response and apoptosis, which showed the highest dysregulation during thermal stress response, and provide a potential physiological basis for both early and late transcriptional regulation (Lewis et al., 2010). the better cultivation and fisheries management of this species at high Proteins are involved in all physiological processes and abnormal temperature. cellular proteins after exposure to stress are considered to be primary indication for initiation of the heat shock response (Sanders, 1990; 2. Materials and methods Lund et al., 2003). Therefore, heat shock proteins have been subjected much attention (Sanders, 1993). Among the heat shock protein groups 2.1. Ethics statement (Hsps), Hsp 70 would probably be the best candidate to indicate thermal stress due to the factor of its sensitivity in response to a large This study was approved by the Institutional Animal Care and Use number of stressors (Liu et al., 2015b). Previous studies displayed that Committee of Heilongjiang River Fisheries Research Institute, Chinese the Hsp level of different tissues in fish exposed to high temperatures Academy of Fishery Sciences. All experimental procedures were in increased when compared with fish maintained under an optimal compliance with the guidelines of University of Guelph on Animal Care. temperature (Currie et al., 2000; Lund et al., 2003). The effects of thermal stress at a proteomic level were also reported in different fish, 2.2. Fish and rearing conditions such as Channa striatus (Mahanty et al., 2016), sturgeon (Acipenser medirostris, Acipenser transmontanus)(Silvestre et al., 2010), goby fish Juvenile lenok were obtained from the Coldwater Fisheries (Gillichthys mirabilis)(Jayasundara et al., 2015). Hatchery Farm at Heilongjiang Fisheries Research Institute, China. The Exposure to an external stressor will result in changes to gene ex- experimental fish (average body weight 90 g) were transferred to 10 pression and protein production, which are amplified at the level of the glass aquariums with electric heaters and water treating equipment metabolome (Lankadurai et al., 2013). Metabonomic approaches re- (60 × 60 × 50 cm, approx. capacity: 125 L) for 4 weeks acclimation at present a novel analytical method investigating small endogenous me- 16 ± 0.5 °C (9 fish/aquarium). During the acclimation, fish were fed to tabolites in complex organisms and their responses to external stimuli, apparent satiation twice daily (09:00 h, 16:00 h) with designed fish which have been employed in many fields of scientific research such as food (Table S1). The fish food was formulated to contain 43% crude nutrition, medicine and toxicology (Cappello et al., 2016; Cappello protein and 12% crude lipid. The feed was made into 2-mm (diameter) et al., 2017; Melis et al., 2017; Cappello et al., 2018; Christopher, pellets, oven-dried at 60 °C and then stored at 4 °C. During the experi- 2018). In particular, non-targeted analysis of NMR-based metabo- ment, the water flow rate through each aquarium was about 600 L/h, nomics, which is a detection of non-specific group of metabolites from a aeration was provided continuously, dissolved oxygen was maintained biological sample, has been used to study the metabolic changes of at above 8 mg/L, pH was approximate 7.5, ammonia-N was < 0.15 mg/ salmonid species exposed to elevated temperatures (Lankadurai et al., L and the photoperiod was set at 12L:12D. 2013). In a previous study, Viant et al. identified a decrease in phos- phocreatine, ATP and glycogen in temperature-exposed steelhead trout 2.3. Experimental design and sampling (Oncorhynchus mykiss); They also provided a more integrated descrip- tion of the biochemical response to thermal stress, such as amino acids, Ten aquariums were randomly assigned as control (16 °C, N = 5) or lactate and phosphocholine (Viant et al., 2003). Metabonomics analyses thermal stress groups (24 °C, N = 5). At the start of the experiment, the of plasma from Atlantic salmon (Salmo salar) revealed changes in en- temperature of stress group was raised to 24 °C using heaters at a ergy metabolism, a decline of several amino acids (glutamine, tyrosine heating rate of 2–3 °C per day. 9 fish from each group were randomly and phenylalanine) and a shift in lipid metabolism under this increased sampled when water temperature of stress group reached 24 °C (day 0), temperature (Kullgren et al., 2013). and 1, 3, 5 and 7 days after it began. During this period, the experi- The lenok (Brachymystax lenok) is a salmonid species with an ori- mental fish of both the control and the stress group were not fed, and ginal distribution in cold freshwater rivers in north-east Eurasia there was no water change for each aquarium to avoid fluctuation of (Alekseyev et al., 2003). It is also one of the few salmonids native to water temperature. To obtain samples, fish were anesthetized with northern China that provides important ecological and economic value metacaine (MS-222) with a dose of 200 mg/L. Blood was collected from (Dong and Jiang, 2008). However, natural populations have decreased caudal vasculature immediately after the fish were anesthetized using a significantly because of over-fishing, habitat degradation and environ- 2.5-ml heparinized syringe, and then the liver was dissected out. mental pollution (Liu et al., 2015a). Fortunately, some investigations Plasma was subsequently separated by centrifugation (4000g for and large-scale artificial reproduction of the lenok have been carried 15 min) at 4 °C and each sample was then aliquoted into two 1.5-ml out for the purpose of conservation and commercialization of this microcentrifuge tubes. One tube was stored at −20 °C for blood species (Mou et al., 2013). The water temperature for optimal growth of chemistry analysis; the other one was immediately frozen in liquid ni- the lenok is 14–18 °C. However, water temperature in culture and in the trogen and stored at −80 °C. One part of each liver was stored in bouin wild where the lenok is found in northern China can reach 24 °C during for 1 day and then moved to ethanol (70%) until histological analysis; summer months and research has already shown that there is a sig- the other part was immediately frozen in liquid nitrogen and stored at nificant decrease in feeding and growth rates at high temperature (Liu, −80 °C until subsequent analysis. 2015). It is therefore important to optimize the cultivation method in order to achieve optimal growth at high temperature. As a first step of 2.4. Histological and blood chemistry analyses achieving such a goal, it is necessary to learn metabolic changes in the lenok when subjecting to high temperature. Liver tissue from 4 fish of each group at the same time point was In this study, the experimental fish were exposed to a sub-lethal collected to investigate the hepatic histological changes in thermal- high temperature (24 °C). Dynamic changes in the metabolic profile stressed fish. Liver tissue was dehydrated with ethanol, and embedded under this high temperature were compared to control (16 °C) using a in paraplast. Sections (7 μm) were stained with hematoxylin and eosin NMR-based metabonomic analysis. High-throughput RNA-Seq was then (H&E), and examined by light microscopy. Standard

309 Y. Liu et al. Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319 spectrophotometric methods for the BECKMAN CX4 automatic analyzer broadening factor for liver extracts prior to Fourier transformation. All were used to measure the following plasmatic parameters: albumin 1H NMR spectra were manually adjusted for phase and baseline dis- (UALB), total protein (TP), triglyceride (TG), cholesterol (CHOL), ala- tortion. The plasma spectra were referenced to the α-glucose at δ 5.23, nine aminotransferase (ALT), aspartate aminotransferase (AST), alka- and those of liver extracts were referenced to the TSMP at δ 0 ppm. The line phosphatase (ALP), lactic dehydrogenase (LDH) and glucose (Glc). spectral region δ 0.3–9 for plasma and δ 0.3–10 for liver extracts were The control and stress groups were compared using the Independent integrated into bins with equal width of 0.004 ppm using the AMIX Samples T Test. Significant change was determined at the level of software (v.3.9.3，Bruker Biospin, Germany). Water regions between δ p < 0.05. 4.40–5.13 for plasma and δ 4.46–5.18 for liver extracts were excluded. Each bucket was normalized to the sum of total integrals for all samples 2.5. HSP70 gene expression prior to statistical analysis. Multivariate data analyses of the normalized NMR data sets were Total RNA was extracted from the livers of 4 fish at each sample carried out using the SIMCAP+ software package (version 12.0, point from the control and the thermal stress group using TRIzol Umetrics, Sweden). The principal component analysis (PCA) was per- Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's formed on mean-centered data to visualize the overview of each data instructions. The isolated RNA was reverse transcribed using a set and find possible outliers (based on the principles of Hotelling's T2). SuperQuickRT cDNA Kit (Toyobo, Shanghai, China), and the obtained Then, projection to latent structures discriminant analysis (PLS-DA) and cDNA was used in subsequent polymerase chain reaction (PCR). Primer orthogonal projection to latent structure discriminant analysis (OPLS- used for qPCR is listed in Table S2. Portions (2 μL) of the synthesized DA) were applied to the analysis of 1H NMR spectral data scaled to unit first-strand cDNA were amplified by PCR in 10 μL reaction mixtures variance with seven-fold cross-validation (Trygg and Wold, 2002; using a BIO-RAD Real-Time PCR System within UltraSYBR Mixture Vandenberg et al., 2006). The model quality was estimated by model (with Rox) kit (CWbio.Co.Ltd.) according to the manufacturer's in- parameter Q2 (indicating the model predictability), permutation test structions. Amplification conditions used for qPCR were 95 °C for (Number = 200) and CV-ANOVA test at the significant level of 10 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 60 s. All p < 0.05 (Eriksson et al., 2008). When the permutation test and the PCR reactions were run in triplicate. Expression levels were calculated CV-ANOVA test were satisfied at the same time, the models were con- according to the threshold cycle (ΔΔCt) method (Livak and Schmittgen, sidered to be valid. To further explain the results of the models, the 2001). loading data after back-transformation (from OPLS-DA) were plotted with the correlation coefficient color-coded for each data point using an 2.6. 1H NMR sample preparation, NMR spectroscopy and NMR data in-house developed Matlab script (MATLAB 7.1, The Mathworks Inc., analysis USA). The color-coded correlation coefficients showed the weights of metabolites contributing to the group separation. A “warm” color (e.g., Plasma samples and liver tissue of 9 fish from the control and the red) corresponds to the metabolite being markedly different between thermal stress groups at each sample point were used for NMR analysis. groups, while a “cool” color (e.g., blue) corresponds to no differences Each plasma sample (30 μL) was mixed with 60 μL 45 mM phosphate between groups. The metabolite was considered to be significantly buffer (pH 7.47, 50% D2O) containing 0.9% NaCl and 400 μLD2O into a different (p < 0.05) between two classes if a correlation coefficient |r| 5 mm NMR tube used directly for NMR detection. is greater than the cutoff value (depending on the sample numbers in Each liver tissue (about 50 mg) was homogenized in cold methanol each group) (Cloarec et al., 2005). and water (2:1, v/v) using a Qiagen Tissue-Lyser (Retsch GmBH, Germany). The supernatant was collected after centrifugation (11,180g, 2.7. RNA extraction, transcriptomic sequencing and differential expression 4 °C, 10 min). The insoluble residues were further extracted twice using analysis the same procedure. The supernatants collected from the above pro- cedure were mixed together and freeze-dried after removal of methanol Total RNA was extracted from 6 fish livers: three from the control, in vacuo. Then the residue was redissolved in 600 μL of phosphate and three from the thermal stress group at day 5 because of the most buffer (0.15 M, pH 7.56, 50% D2O) containing 0.001% TSP and 0.01% significant metabolic changes. TRIzol Reagent (Invitrogen, Carlsbad, NaN3. The supernatant (550 μL) was transferred into a 5 mm NMR tube CA, USA) was used following the manufacturer's instructions. A total ready for NMR analysis. amount of 2 μg RNA per sample was used as input material for the RNA All NMR spectra were recorded at 298 k on a Bruker Avance III sample preparations. Sequencing libraries were generated using 600 MHz NMR spectrometer (600.13 MHz for proton frequency) NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, equipped with a cryogenic probe (Bruker Biospin, Germany). 1H NMR USA) following the manufacturer's recommendations and index codes spectra of plasma were acquired using the Carr-Purcell-Meiboom-Gill were added to attribute sequences to each sample. The RNA con-

(CPMG) pulse sequence [RD-90°-(τ-180°-τ)n-ACQ] with water pre- centration of library was measured using Qubit® RNA Assay Kit in saturation, where τ = 350 us and n = 100. The total transverse re- Qubit® 3.0 to preliminary quantity and then diluted to 1 ng/μL. Insert laxation delay of 70 ms was used. For liver extract samples, the NOESY size was assessed using the Agilent Bioanalyzer 2100 system (Agilent pulse sequence [RD-90°-t1-90°-tm-90°-acquisition] was used with water Technologies, CA, USA), and the qualified insert size was accurate presaturation during the recycle delay (2 s) and mixing time (80 ms). A quantification using StepOnePlus™ Real-Time PCR System (Library total of 128 transients for all samples were collected into 32 k data valid concentration > 10 nM). The clustering of the index-coded points over a spectral width of 20 ppm with a 90° pulse length adjusted samples was performed on a cBot cluster generation system using HiSeq to about 10 μs. For assignment purposes, two-dimensional NMR spectra, PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer's including 1He1H total correlation spectroscopy (TOCSY), 1He1H cor- instructions. After cluster generation, the libraries were sequenced on relation spectroscopy (COSY), 1He1H J-resolved spectroscopy (JRES), an Illumina Hiseq 4000 platform and 150 bp paired-end reads were 1He13C heteronuclear single quantum coherence spectroscopy (HSQC) generated. and 1He13C heteronuclear multiple bond correlation spectroscopy Clean reads were gained by removing adaptor-only reads, repeated (HMBC), were recorded and processed on the same NMR spectrometer reads and low-quality reads. Contig assembly was carried out using the in a similar way as reported previously with slightly changed para- short read assembling program Trinity (the default options and a meters (Dai et al., 2010). minimum allowed length of 200 bp) with 100 GB of memory and a path An exponential function was applied to all free induction decays reinforcement distance of 50. Then, the contigs were clustered by the with a 1 Hz line-broadening factor for plasma and 0.5 Hz line- sequence clustering software TGICL to obtain unigenes. The assembled

310 Y. Liu et al. Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319 unigenes were annotated by homology searches against NCBI nr the remaining samples were further analyzed using PLS-DA and OPLS- (http://www.ncbi.nlm.nih.gov/), Swissprot (http://www.expasy.ch/ DA and the model quality was assessed by the use of Q2, permutation sprot/) and the KEGG pathway (http://www.genome.jp/kegg/) data- test and CV-ANOVA test. As seen in Table S12, the results showed that bases with BLAST program (E-value < 1E−5). We also utilized the with the exception of the first time point (C0 vs. TS0) from liver, each Blast2GO program for the functional annotation of unigenes. model displayed a difference in its metabolic profiles. The corre- DEGseq v1.18.0 was used for differential gene expression analysis sponding score plots and their back-scaled loading plots of the sig- between two groups with biological replicates. Under the assumption nificant models (from OPLS-DA) were shown in Fig. 4 and Fig. 5, where that the number of reads derived from a gene (or transcript isoform) “warm” colors (e.g., red) corresponded to the metabolites being sig- follows a binomial distribution, DEGseq was proposed based on the MA- nificantly different between two groups. “Up” and “down” of signals plot and is widely used for differential gene expression analysis. Genes indicated relatively increased or decreased of metabolites. The key with p ≤ 0.05 and |log2_ratio| ≥ 1 were identified as differentially metabolites associated with thermal stress in plasma and liver extracts expressed genes (DEGs). DEGs were also used in pathway enrichment were illustrated in Fig. 6. In comparison with the control group, marked analysis. GOstat and KOBAS were applied to perform GO and KEGG changes in the stress group were mainly shown by an increase in lipid enrichment analysis. The hypergeometric test p-value ≤ 0.01 was con- status. These changes included elevated levels of PUFA (δ2.78 ppm), sidered significant for all analyses. UFA (δ5.30 ppm), ω3PUFA (δ0.93 ppm) and lipids (δ2.30 ppm) throughout the experimental period with an exception of a reduction in 3. Results the level of lipids (δ1.96 ppm) at days 0, 1, 3 and 5, as well as a decrease in the level of HDL (δ0.83 ppm) at days 0, 3, 5 and 7. There was also a 3.1. Histology and blood chemistry analysis significant reduction in the level of inosine in the stress group compared to the control group. In addition, the decreased concentrations of gly- Histological analysis of liver tissue of the lenok from the control (C) cine and dimethylglycine (days 0, 3, 5 and 7), glycerophosphoryl and the thermal stress (TS) groups at different time points were shown choline and uridine (days 0, 3, 5 and 7) were observed in the stress in Fig. S1. Normal cell systems of liver were observed in both control group compared to the control group. Increased levels of valine and group and stress group at day 0 (Fig. S1, TS0). Hepatic autolysis of liver glutamine at days 3 and 5 were also observed in the stress group. cells of fish in the stress group began to appear at day 1 (Fig. S1, TS1) In liver, predominant variations were shown by an increase in and hazy cell membrane of fish liver in stress group was observed at day glutamine and uridine diphosphate levels as well as a reduction of 3 of heating (Fig. S1, TS3). Lipid droplets and widespread disintegrated taurine and lysine levels at days 1, 3, 5 and 7 in stress group, compared cell systems were observed in the stress group at days 5 and 7 with to control group. The concentrations of leucine, valine, arginine, phe- much more severity at day 7 than day 5 (Fig. S1, TS5 and TS7). This nylalanine and choline in the stress group were decreased significantly result suggested that longer term thermal stress caused hepatic stea- at days 3, 5 and 7. Moreover, the levels of alanine and glycine were also tosis. decreased at days 5 and 7. The level of glutamate in the stress group The thermal-induced changes in blood biochemistry in the lenok increased dramatically at days 3, 5 and 7. were shown in Fig. 1. More changes in blood biochemistry were ob- served as the temperature was increased. The thermal stress induced 3.4. Transcriptomic profiling elevations in the levels of ALT, CHOL and LDH at days 1, 3, 5 and 7; the levels of UALB and AST at days 3, 5 and 7; the levels of TP, Glc, TG at To validate the metabolic changes induced by thermal stress, liver days 5 and 7 (p < 0.05). However, the level of ALP was decreased at samples at day 5 were selected to compare gene expression profiles days 3, 5 and 7 in the thermal stress group compared to the control between the stress group and the control group using RNA-Seq. This is group (p < 0.05). because the most significantly metabonomic changes were observed at this time point. De novo assemblies of all libraries resulted in a total of 3.2. Hsp70 mRNA expression 235,397 unigenes, among which, 93,330, 57,584, 47,051 and 47,712 unigenes were annotated in NCBI non-redundant (nr), Swissprot, GO The mRNA expression of Hsp70 in the liver was up-regulated ra- and KEGG pathway databases, respectively (Table S3, S4; Fig. S2). pidly at day 0 and reached its highest value (15-fold, Fig. 2) and then A total of 2986 genes were differentially expressed between the gradually declined as the fish were subjected to prolonged thermal control and the stress group, of which 1448 genes were significantly up- stress. No significant change was observed in the expression levels of regulated while 1538 genes were significantly down-regulated under Hsp70 at day 7 between the control and the stress group. thermal stress (|log2Ratio| ≥ 1 and p < 0.01; Fig. S3). 1448 up-regu- lated genes were mapped to 14 pathways and 1538 down-regulated 3.3. NMR spectroscopy of plasma and liver tissue and multivariate data genes were mapped to 21 pathways in KEGG including energy meta- analysis bolism, lipid metabolism, amino acid metabolism and nucleotide me- tabolism (p < 0.01; Table S5, S6). The typical 1H NMR spectra of plasma and liver extracts in the control and thermal stress group were shown in Fig. 3. The metabolites 3.5. Analysis of differential expression genes (DEGS) between the control were assigned according to literature data, extensive 2D NMR spec- and the stress group troscopy and statistical total correlation spectroscopy (STOCSY) (Nicholson et al., 1995; Coen et al., 2003; Yap et al., 2006). Plasma Following thermal stress treatment, the expression levels of genes spectra were dominated by amino acids, glucose, glycerophosphoryl related to energy metabolism were significantly changed. Some of the choline, nucleotides, organic acids, acetate, lipoprotein and fatty acids, genes coding for proteins, such as CA4 related to nitrogen metabolism, while the NMR spectra of liver extracts were mainly comprised of or- GCK, and G6PC related to glycolysis/gluconeogenesis, were sig- ganic acids, nucleotides, glycogen, amino acids, acetate, taurine, cho- nificantly down-regulated. In contrast, the expression levels of some line and lipid. The detailed assignments of metabolites from plasma and other genes coding for proteins, such as ACO related to TCA cycle, GPI, liver extracts were shown in Table S15. The integral data of 1H NMR HK, FBP and GAPDHS related to glycolysis/gluconeogenesis were sig- spectra of plasma and liver extracts were analyzed by PCA, PLS-DA and nificantly increased (Table S7). OPLS-DA. Based on the 95% confidence limit, several samples (7 Under thermal stress treatment, there was a general increase in the plasma samples and 3 liver extracts samples) were identified as outliers expression of genes related to lipid metabolisms, such as alpha-linolenic and subsequently excluded owing to the bad water suppression. Thus, acid metabolism, linoleic acid metabolism, ether lipid metabolism and

311 Y. Liu et al. Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319

14.00 60.00 25.00 CTS * * CTS CTS 12.00 * 50.00 * 20.00 * 10.00 * *

* )L/UI(TLA L/g 40.00 ) ( 8.00 * L/(P 15.00 B * 30.00 * LAU *

6.00 T 10.00 4.00 20.00 2.00 10.00 5.00 0.00 0.00 0.00 d0 d1 d3 d5 d7 d0 d1 d3 d5 d7 d0 d1 d3 d5 d7 Sampling time(d) Sampling time(d) Sampling time(d)

600.00 CTS 140.00 8.00 CTS * CTS* 500.00 120.00 7.00 )L/UI(TSA

)L/lomm(GT 6.00 400.00 )L 100.00 / * 5.00 UI(P 80.00 * 300.00 * 4.00 60.00 LA * 200.00 3.00 40.00 100.00 2.00 20.00 1.00 0.00 0.00 0.00 d0 d1 d3 d5 d7 d0 d1 d3 d5 d7 d0 d1 d3 d5 d7 Sampling time(d) Sampling time(d) Sampling time(d)

3000.00 12.00 CTS 20.00 * CTS* CTS * 18.00 * 2500.00 10.00

)L/lomm(LOHC 16.00 )L/l 14.00 )L/UI(HDL 2000.00 8.00 12.00 o mm(clG 10.00 1500.00 6.00 8.00 1000.00 4.00 6.00 4.00 500.00 2.00 2.00 0.00 0.00 0.00 d0 d1 d3 d5 d7 d0 d1 d3 d5 d7 d0 d1 d3 d5 d7

Sampling time(d) Sampling time(d) Sampling time(d)

Fig. 1. Effect of thermal stress on selected blood chemistry parameters in the lenok. C, Control group; TS, Thermal stress group; d, day UALB, albumin; TP, total protein; TG, triglyceride; CHOL, cholesterol; ALT, aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; LDH, lactic dehydrogenase; Glc, glucose

25.0 was a significant change in the expression of genes related to heat shock

slevelANRm protein including increased Hsp70 and decreased Hsp30 (Table S11). 20.0 C TS

15.0 4. Discussion

ev 10.0

it 4.1. HSP70 gene expression a leR 5.0 Heat shock proteins (hsp), which act as molecular chaperones to re- 0.0 fold thermally damaged proteins and prevent their cytotoxic aggrega- d0 d1 d3 d5 d7 tion, are commonly used indicators to assess the effects of thermal stress Sampling time(d) on fish (Dalvi et al., 2012). Among them, Hsp70 is the most commonly Fig. 2. Relative mRNA levels of Hsp70 in liver of the lenok exposed to thermal expressed heat shock protein in response to elevated temperatures stress. (Fowler et al., 2009). In this study, the expression level of Hsp70 in the C, Control group; TS, Thermal stress group; d, day liver was up-regulated after exposure to high temperature, but no change was observed when subjected to prolonged thermal stress. Transcriptomic results also showed that there was a significant increase arachidonic acid metabolism. These genes coded for proteins, such as in Hsp70 at day 5. Previous studies revealed that Hsp70 mRNA was Gpx, CYP21A, FASN, E3.1.1.3, PLA2G, FadD, PHS1, GLB1 and PLA2G4 markedly up-regulated following acute temperature stress, and then (Table S8). For amino acid metabolism, there was an increase in genes leveling-off was observed as stress was prolonged (Lund et al., 2003; coded for lysine degradation (PLOD1, NSD1 and MLL3), valine, leucine Lewis et al., 2010). The expression of Hsp70 returning to its normal and isoleucine degradation (IVD and DBT), arginine biosynthesis level after relatively long-term thermal acclimation has been postulated (E3.5.3.1) and D-glutamine and D-glutamate metabolism (GLS). We also to be due to the completion of synthesizing enough protein to re-fold observed a decrease in genes encoding protein CSAD in taurine and thermally damaged proteins under high temperature (Buckley et al., hypotaurine metabolism pathways (Table S9). The expression levels of 2006; Logan and Buckley, 2015). Our results suggested that liver da- genes related to nucleotide metabolism were significantly decreased mage could be responsible for the observed leveling-off of the expres- following thermal stress treatment. These genes were coded for pro- sion of Hsp70 after a prolonged thermal stress, which can be verified by teins, including purA, punA and ADCY10 (Table S10). In addition, there blood chemistry and histology. In the present study, the histological

312 Y. Liu et al. Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319

a) C 8* p9 26 p8 p4 p3 27 24 p6 29 28 35 13 9 7 33 33 4 17 8,9 plasma

TS 36 5 36 p2 36 p7 5 22 glc p5 38 p1 36 14

5.56.06.57.07.58.08.5 1.01.52.02.53.03.54.04.5 ppm b) C 8* 40 32 40 31 11,12 33 33 31 21 12 16 1,2,3 30 30 31 8 liver

25 15 TS 7 29 28 23 5,6 37 34 37 23 20 11 39 34 41 32 18 18 10 p3 p2 19

5.56.06.57.07.58.08.5 1.01.52.02.53.03.54.04.5 ppm

Fig. 3. 1H NMR spectra of plasma (a) and liver extracts (b), and metabolites assignment of the lenok. C, Control group; TS, Thermal stress group; 1, valine; 2, leucine; 3, isoleucine; 4, ethanol; 5, lactate; 6, threonine; 7, alanine; 8, lysine; 9, arginine; 10, acetate; 11, glutamine; 12, glutamate; 13, methionine; 14, pyruvate; 15, succinate; 16, asparatate; 17, citrate; 18, β-alanine; 19, malate; 20, TMA; 21, dimethylglycine; 22, creatine; 23, choline; 24, GPC; 25, taurine; 26, betaine; 27, glycine; 28, β-glucose; 29, α-glucose; 30, uracil; 31, uridine; 32, fumarate; 33, tyrosine; 34, histidine; 35, phenylalanine; 36, inosine; 37, nicotinamide; 38, hypoxanthine; 39, formate; 40, UDP; 41, glycogen; p1, cholesterol; p2, lipids (HDL, LDL, VLDL); p3, lipids(ω3 fatty acid); p4, lipids(HDL, LDL, VLDL); p5, lipids; p6, unsatured fatty acids; p7, lipids; p8, polyunsatured fatty acids; p9, unsatured fatty acids. results clearly showed that high temperature resulted in extensive lipid gluconeogenesis were significantly down-regulated at day 5 (Table S7). droplet accumulation and structural alterations of the hepatocytes as Therefore, the energy metabolism in the current study was obviously stress time was prolonged. Furthermore, there was a significant in- disturbed by thermal stress. In agreement, mobilization of energy crease in the levels of blood ALT and AST compared to the control substrates by fish under stress condition has also been reported in a group. Therefore, it is questionable if Hsp 70 mRNA expression could be previous study (Brandão et al., 2015). a biomarker for chronic high temperature stress. In the present study, the increased lipids in plasma, including un- saturated fatty acids, cholesterol and triglyceride, indicated that there 4.2. Energy and lipid metabolism was a shift in lipid metabolism. Additionally, in the liver transcriptome of the lenok following thermal stress at day 5, some genes and pathways It is well known that the physiological compensation to stressors is related to lipid metabolism were significantly up-regulated (S7, S10). an energy-demanding process that requires the fish to mobilize energy These results were in accordance with a previous study on Atlantic substrates to cope with stress (Barton and Schreck, 1987; Costas et al., salmon showing that there were increased plasma VLDL and lipids 2011; Zhou et al., 2011). In our study, alterations in the levels of citric containing unsaturated FA (Kullgren et al., 2013). Since hepatic stea- acid and succinate, which are key intermediate products of the tri- tosis was also observed in stressed fish, the increased lipids in plasma carboxylic acid (TCA) cycle, were detected. There was an elevated level may be partially due to mitigating lipid accumulation in the liver and of succinate in the liver at day 1 and in plasma at days 0 and 1, which maintaining homeostasis in fish at high temperature. Supportive evi- indicated an energy demand of the lenok following high temperature dence also comes from the decreased HDL level and increased VLDL in exposure. However, citric acid concentration in plasma of the stress plasma (Fig. 2). group showed a transient increase at day 1 and a decrease at days 5 and 7 relative to the control group, which implied that this could be an early 4.3. Amino acid metabolism response to recruit energy as a temperature elevation, and then was repressed with prolonged thermal stress (Fig. 6). Concomitantly, tran- It has been reported that high temperature could result in an ele- scriptomic results showed that some important genes related to energy vated ammonia level in fish, which is produced from the catabolism of metabolism such as nitrogen metabolism and glycolysis/ proteins and amino acids in the liver (Ballantyne, 2001; Currie et al.,

313 Y. Liu et al. Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319

(caption on next page)

314 Y. Liu et al. Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319

Fig. 4. OPLS-DA score plots (left) and corresponding coefficient-coded loading plots (right) obtained from metabolic profiles of 1H NMR spectra of the plasma samples. Upright signals indicate a relatively increased intensity of metabolites, and downright peaks a decreased intensity of metabolites. The colors shown on the plot are associated with the significance of metabolites in separating the samples; red indicating significance at a level of p < 0.05. C, Control group; TS, Thermal stress group; Sample number for analyses: C0, 8; TS0, 8; C1, 8; TS1, 9; C3, 8; TS3, 7; C5, 9; TS5, 9; C7, 9; TS7, 9. Glc glucose, Gln glutamine, Gly glycine, GPC glycerophosphorylcholine, Urid uridine, Inos inosine, DMG dimethylglycine, Ci citrate, Succ succinate, Ace acetate, UFA unsatured fatty acids, PUFA polyunsatured fatty acids, ω3PUFA ω3 polyunsatured fatty acids, lipids (2.30↑, 1.96↓), HDL high-density lipoprotein, VLDL very low density lipoprotein.

Fig. 5. OPLS-DA score plots (left) and corresponding coefficient-coded loading plots (right) obtained from metabolic profiles of 1H NMR spectra of the liver extracts samples. Upright signals indicate a relatively increased intensity of metabolites, and downright peaks a decreased intensity of metabolites. The colors shown on the plot are associated with the significance of metabolites in separating the samples; red indicating significance at a level of p < 0.05. C, Control group; TS, Thermal stress group; Sample number for analyses: C1, 7; TS1, 9; C3, 9; TS3, 8; C5, 8; TS5, 8; C7, 7; TS7, 9. GPC glycerophosphorylcholine, PC phosphorylcholine, UDP uridine diphosphate, Glu glutamate, Gln glutamine, Gly glycine, tau taurine, β-ala β-alanine, lys lysine, phe phenylalanine, tyr tyrosine, arg arginine, val valine, leu leucine, ileu isoleucine, asp asparatate, his histidine, lipid lipids (2.30↑, 1.96↓).

315 Y. Liu et al. Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319

Fig. 6. Dynamic liver extract and plasma metabonomic changes for the Lenok in the thermal stress (TS) group and the control (TS) groups. The heatmaps were generated from corre- lation coefficients of metabolite changes as a function of sample time (i.e., days in horizontal axis) with warm (red) colored bars denoting sta- tistical significant elevation, cold co- lored (blue) ones indicating decreases compared with control groups, and white ones indicating no statistical significance.

2010). In our study, many amino acids in the liver and plasma in the proteins, taurine is still considered a conditionally essential amino acid stress group had a significant decrease relative to the control group, and is involved in many physiological functions in fish, including os- which implied that there could be an accumulation of body ammonia motic regulation, antioxidation, feeding stimulation and bile salt con- due to amino acid catabolism. As ammonia is a neurotoxin, high tissue jugation (El-Sayed, 2013; Brandão et al., 2015; Salze and Davis, 2015). ammonia concentration is harmful to fish health (Tsui et al., 2002). In The conjugation of taurine and free bile acids in fish was taurocholic order to survive, fish exposed to a high temperature need to cope with a acid, which may enhance lipid metabolism in fish, through the increase high level of body ammonia. Glutamate and glutamine are non-essen- in the activity of the bile salt-activated lipase in the liver (Chatzifotis tial amino acids (NEAA) in fish, and glutamine can be synthesized from et al., 2008; El-Sayed, 2013). In the present study, hepatic steatosis was glutamate by glutamine synthetase and converted to glutamate by observed in the stressed fish, and there was a depleted concentration of glutaminase. In particular, glutamate metabolism in fish is primarily taurine in the liver both at the metabonomic and transcriptomic levels. deaminated with the production of ammonia due to amino acid cata- As no significant change was observed in other sulfur amino acids, the bolism, which differs from that of mammals (Ballantyne, 2001). In the decreased taurine may be due to lipid digestion and the formation of present study, unlike most other amino acids, glutamate and glutamine bile salts. Similar findings have been reported that taurine caused a levels showed a significant increase, which was different from previous decrease of lipids in the liver to improve hepatic steatosis in zebrafish findings in salmonid species (e.g., Atlantic salmon and steelhead trout) (Danio rerio) induced by thioacetamide (Hammes et al., 2012). (Viant et al., 2003; Kullgren et al., 2013). The possible reason for this Whereas, a lack of taurine in the diet may also have negative effects on discrepancy could be the higher water temperature of the stress group the function of liver and immune system in fish (Kim et al., 2007). in the current study (24 °C) compared with 18 °C and 20 °C in previous studies. Therefore, our present results imply that the continuously 4.4. Choline metabolism and nucleotide metabolism elevated level of glutamate and glutamine could be an important way to reduce ammonia accumulation and weaken its toxicity for the lenok Thermal stress caused a reduction in the levels of choline and gly- under thermal stress. Results from transcriptomic changes showed that cerophosphoryl choline, indicating alterations in choline metabolism. there was a significant increase in glutaminase, which confirmed that This result was in line with a previous study on Atlantic salmon sub- glutamate metabolism may play an important role in resistance to jected to elevated temperature (Kullgren et al., 2013). Choline is an thermal stress for the lenok. Supporting evidence also comes from the essential constituent of cell membrane. It is a lipotropic agent that observation of a lasting drop of lysine in the liver both at metabonomic prevents excessive lipid accumulation in the liver by promoting the and transcriptomic levels. Catabolism of lysine was highly affected by removal of triacylglycerols from hepatocytes via their incorporation stress since lysine could be converted to glutamate and other stress- into lipoproteins (Pinotti et al., 2002). In the present study, thermal related metabolites in response to stress (Galili et al., 2001). Moreover, stress induced significant lipid shift and lipoprotein alterations in as a constituent of glutathione, glutamate may play an important role in plasma including depleted HDL and increased VLDL, which suggest that the activation of antioxidative defense. It has been reported that dietary choline could play an important role in lipid transport to mitigate lipid glutamine supplementation could promote the health and survival of accumulation in the liver under high temperature. fish through enhancing antioxidant abilities and stress resistance (Liu Nucleotides and their related metabolic products play key roles in et al., 2015c). Therefore, based on the above analyses, we hypothesis many biological processes，which have many metabolic functions in- glutamate may be a very important biomarker associated with thermal cluding mediating energy metabolism as well as serving as nucleic acid stress. Further study is needed to investigate how exogenous glutamate precursors, physiological mediators, components of coenzymes, and or glutamine in the diet regulates the lenok to adapt to thermal stress. activated intermediates (Carver and Walker, 1995). Purine nucleosides, Taurine, one of the most abundant free amino acids in vertebrates, such as adenosine and its primary metabolite inosine, are low-mole- is synthesized endogenously from sulfur amino acids such as methio- cular-weight molecules that participate in a wide variety of in- nine and cysteine (Huxtable, 1992). Although not incorporated into tracellular biochemical processes and serve as monomeric precursors of

316 Y. Liu et al. Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319

RNA and DNA (Haskó et al., 2004). Uridine is a pyrimidine nucleoside maintaining homeostasis in fish at high temperature. Changes in cho- that plays a crucial role in the synthesis of RNA, glycogen, and the line metabolism could play an important role in lipid transport to mi- biomembrane (Yamamoto et al., 2011). In the present study, the inosine tigate lipid accumulation in the liver under a high temperature. The and uridine levels decreased in the thermal stress group relative to the decreased inosine and uridine level indicated that the synthetic rate of control group. Transcriptomic results showed that there was a decrease DNA, RNA, and protein in experimental fish could be reduced by high in adenylosuccinate synthase, purine-nucleoside phosphorylase and temperature. Results from transcriptomic changes showed activations adenylate cyclase 10 at day 5. These results indicated that the synthetic of genes regulating energy metabolism, lipid metabolism and nucleo- rate of DNA, RNA, and protein in experimental fish could be reduced by tide metabolism. Evidence from Hsp 70 gene expression, blood bio- high temperature. Little is known about nucleotides in response to chemistry and histology also confirmed that high temperature exposure thermal stress. However, it has been reported that dietary nucleotide had negative effects on the health of this fish species. These results supplementation enhanced growth and immune responses of grouper provide a multi-faceted view of the response of the lenok to thermal Epinephelus malabaricus (Lin et al., 2009). Dietary administrations of stress. mixed nucleotides promotes growth, immune responses and stress re- In the present paper, metabolic changes of fish were compared in sistance of juvenile red sea bream Pagrus major (Hossain et al., 2016). the control and stress groups at the same time point to better under- Therefore, the variation of nucleotides in this study supported the no- stand the metabolic mechanisms of the lenok to thermal stress. All the tion that sufficient dietary nucleotide supplementation may also be experimental fish were not fed during the course of experiment to keep necessary in the lenok against thermal stress. culture conditions in the control and stress groups consistent except The present study builds up evidence of thermal stress effects at water temperature, because feed intake of juvenile lenok was seldom different levels on the lenok. The results from metabonomic and tran- happening at a high temperature of 24 °C. If the fish were fed, food can scriptomic analyses indicated that thermal stress caused complex me- be another factor which influence metabolism in the lenok dramati- tabolic changes from multiple related metabolic pathways at the sys- cally. Furthermore, results from previous study showed that short-term tems level (Fig. 7). Such changes involved transamination, TCA cycle, starvation has minor effects on fish health and stress response (Tian lipid metabolism, amino acid metabolism, choline metabolism and et al., 2010; Liu et al., 2011; Waagbø et al., 2017). More research is nucleotide metabolic processes. Specifically, thermal stress induced an needed to validate the changes in metabolites and investigate nutrition activation of glutamate metabolism, which could be a rapid and ef- control for lighting thermal stress for the lenok. fective way to reduce ammonia accumulation and weaken its toxicity to the lenok exposed to high water temperature. Results from tran- scriptomic changes showed that there was a significant increase in 5. Conclusions glutaminase, which confirmed that glutamate metabolism may play an fi important role in resistance to thermal stress for the lenok. Alterations This study showed that thermal stress not only induced a signi cant in the levels of several key intermediate products of the tricarboxylic increase in Hsp70 and histopathological changes, but also caused sig- fi ff fi acid (TCA) cycle indicated that the fish need to mobilize energy sub- ni cant di erences in metabolic pro les. The dynamic metabolic re- strates (e.g. amino acids) to cope with stress. A shift in lipid metabolism sponses of the lenok to thermal stress associated with the repression of may be partially due to mitigating lipid accumulation in the liver and energy metabolism, shifts in lipid metabolism, catabolism of amino acids, biosynthesis of glutamate and glutamine together with

Fig. 7. Altered metabolic pathways induced by thermal stress at different time points. Red symbols denoted significant increases (p < 0.05) and green ones denoted significant decreases (p < 0.05).

317 Y. Liu et al. Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319 alterations in choline and nucleotide metabolisms. Glutamate may be a Afonso, A., 2011. Physiological responses of Senegalese sole (Solea senegalensis Kaup, very important biomarker associated with thermal stress. The results 1858) after stress challenge: effects on non-specific immune parameters, plasma free amino acids and energy metabolism. Aquaculture 316, 68–76. provide detailed metabolic information and demonstrate that in- Currie, S., Moyes, C.D., Tufts, B.L., 2000. The effects of heat shock and acclimation tegrated analyses, combining the NMR-based metabonomics and tran- temperature on Hsp70 and Hsp30 mRNA expression in trout: in vivo and in vitro scriptome strategy, is a powerful approach to understand the response comparisons. J. Fish Biol. 56, 398–408. fi Currie, S., Bagatto, B., DeMille, M., Learner, A., LeBlanc, D., Marks, C., Ong, K., Parker, J., mechanisms of the lenok to thermal stress. These ndings also imply Templeman, N., Tufts, B.L., Wright, P.A., 2010. Metabolism, nitrogen excretion, and that a high temperature of 24 °C has certainly had an adverse effect on heat shock proteins in the central mudminnow (Umbra limi), a facultative air- the health of the lenok. Further work is essential to study how to ease breathing fish living in a variable environment. Can. J. Zool. 88, 43–58. the negative impacts on the health of cold water fish exposed to high Dai, H., Xiao, C.N., Liu, H.B., Hao, F.H., Tang, H.R., 2010. The combined NMR and LC- DAD-MS analysis reveals comprehensive metabonomic variations for three pheno- temperature. typic cultivars of Salvia Miltiorrhiza Bunge. J. Proteome Res. 9, 1565–1578. Dalvi, R.S., Pal, A.K., Tiwari, L.R., Baruah, K., 2012. Influence of acclimation temperature fi Conflict of interest on the induction of heat-shock protein 70 in the cat sh Horabagrus brachysoma (Günther). Fish Physiol. Biochem. 38, 919–927. Dong, C., Jiang, Z., 2008. Fisheries Resources of Cold Water Fish in the Interior of China. All authors declare that they have no conflict of interest. Heilongjiang Science and Technology Press, Harbin (in Chinese). El-Sayed, A.M., 2013. Is dietary taurine supplementation beneficial for farmed fish and shrimp? A comprehensive review. Rev. Aquac. 5, 1–15. Notes Eriksson, L., Trygg, J., Wold, S., 2008. CV-ANOVA for significance testing of PLS and OPLS(R) models. J. Chemom. 22 (11−12), 594–600. The authors declare no competing financial interest. Fang, J., Tian, X., Dong, S., 2010. The influence of water temperature and ration on the growth, body composition and energy budget of tongue sole (Cynoglossus semilaevis). Aquaculture 299, 106–114. Acknowledgements Fowler, S.L., Hamilton, D., Currie, S.A., 2009. Comparison of the heat shock response in juvenile and adult rainbow trout (Oncorhynchus mykiss) –implications for increased thermal sensitivity with age. Can. J. Fish. Aquat. Sci. 66 (1), 91–100. This study was supported by National Natural Science Foundation of Galili, G., Tang, G.L., Zhu, X.H., Gakiere, B., 2001. Lysine catabolism: a stress and de- China (Grant No. 31502188, 21205130), China Agriculture Research velopment super-regulated metabolic pathway. Curr. Opin. Plant Biol. 4, 261–266. System (Grant No. CARS-46), Natural Science Foundation of Hammes, T.O., Pedroso, G.L., Hartmann, C.R., Escobar, T.D.C., Fracasso, L.B., Rosa, D.P., ff Heilongjiang Province of China (Grant No. QC2015040), Central–Level Marroni, N.P., Porawski, M., Silveira, T.R., 2012. The e ect of taurine on hepatic steatosis induced by thioacetamide in zebrafish (Danio rerio). Dig. Dis. Sci. 57, Non–profit Scientific Research Institutes Special Funds in China (Grant 675–682. No. HSY201602) and China Scholarship Council (Grant No. Haskó, G., Michail, V.S., Szabo, C., 2004. Immunomodulatory and neuroprotective effects – 201703260016). of inosine. Trends Pharmacol. Sci. 25 (3), 152 157. Hossain, M.S., Koshio, S., Ishikawa, M., Yokoyama, S., Sony, N.M., 2016. Dietary nu- cleotide administration influences growth, immune responses and oxidative stress Appendix A. Supplementary data resistance of juvenile red sea bream (Pagrus major). Aquaculture 455, 41–49. Huxtable, R.J., 1992. Physiological action of taurine. Phys. Rev. 72, 101–163. Jayasundara, N., Tomanek, L., Dowd, W.W., Somero1, G.N., 2015. Proteomic analysis of Supplementary data to this article can be found online at https:// cardiac response to thermal acclimation in the eurythermal goby fish Gillichthys doi.org/10.1016/j.cbd.2019.01.006. mirabilis. J. Exp. Biol. 218, 1359–1372. Jeffries, K.M., Hinch, S.G., Sierocinski, T., Pavlidis, P., Miller, K.M., 2014. Transcriptomic responses to high water temperature in two species of Pacific salmon. Evol. Appl. 7, References 286–300. Kim, S.-K., Matsunari, H., Takeuchi, T., Yokoyama, M., Murata, Y., Ishihara, K., 2007. ff ff Alekseyev, S.S., Kirillov, A.F., Samusenok, V.P., 2003. Distribution and morphology of the E ect of di erent dietary taurine levels on the conjugated bile acid composition and fi fl sharp-snouted and the blunt-snouted lenoks of the genus Brachymystax (Salmonidae) growth performance of juvenile and ngerling Japanese ounder Paralichthys oliva- – of East Siberia. J. Ichthyol. 43, 350–373. ceus. Aquaculture 273, 595 601. Ballantyne, J.S., 2001. Amino acid metabolism. Fish Phys. 20, 77–107. Kullgren, A., Fredrik Jutfelt, F., Fontanillas, R., Sundell, K., Samuelsson, L., Wiklander, K., Barton, B.A., Schreck, C.B., 1987. Metabolic cost of acute physical stress in juvenile Kling, P., Koppe, W., Larsson, D.G., Björnsson, B.T., Jönsson, E., 2013. The impact of steelhead. Trans. Am. Fish. Soc. 116, 257–263. temperature on the metabolome and endocrine metabolic signals in Atlantic salmon – Brandão, F., Cappello, T., Raimundo, J., Santos, M.A., Maisano, M., Mauceri, A., Pacheco, (Salmo salar). Comp. Bioc. Physiol. A 164, 44 53. M., Pereira, P., 2015. Unravelling the mechanisms of mercury hepatotoxicity in wild Lankadurai, B.P., Nagato, E.G., Simpson, M.J., 2013. Environmental metabolomics: an fish (Liza aurata) through a triad approach: bioaccumulation, metabolomic profiles emerging approach to study organism responses to environmental stressors. Environ. – and oxidative stress. Metallomics 7 (9), 1352–1363. Rev. 21, 180 205. Buckley, B.A., Gracey, A.Y., Somero, G.N., 2006. The cellular response to heat stress in the Lewis, J.M., Hori, T.S., Rise, M.L., Walsh, P.J., Currie, S., 2010. Transcriptome responses goby Gillichthys mirabilis: a cDNA microarray and protein-level analysis. J. Exp. Biol. to heat stress in the nucleated red blood cells of the rainbow trout (Oncorhynchus – 209, 2660–2677. mykiss). Physiol. Genomics 42, 361 373. Cappello, T., Pereira, P., Maisano, M., Mauceri, A., Pacheco, M., Fasulo, S., 2016. Lin, Y., Wang, H., Shiau, S., 2009. Dietary nucleotide supplementation enhances growth – Advances in understanding the mechanisms of mercury toxicity in wild golden grey and immune responses of grouper, Epinephelus malabaricus. Aqua. Nutri. 15, 117 122. mullet (Liza aurata)by1H NMR-based metabolomics. Environ. Pollut. 219, 139–148. Liu, Y., 2015. Studies on the Energy Budgets and Feeding System for the Lenok Cappello, T., Fernandes, D., Maisano, M., Casano, A., Bonastre, M., Mauceri, A., Fasulo, (Brachymystax lenok)[D]. Institute of Hydrobiology, Chinese Academy of Sciences, S., Porte, C., 2017. Sex steroids and metabolic responses in mussels Mytilus gallo- Wuhan. provincialis exposed to drospirenone. Ecotoxicol. Environ. Saf. 143, 166–172. Liu, W., Wei, Q.W., Wen, H., Jiang, M., Wu, F., Shi, Y., 2011. Compensatory growth in ff Cappello, T., Giannetto, A., Parrino, V., Marco, G.D., Mauceri, A., Maisano, M., 2018. juvenile Chinese sturgeon (Acipenser sinensis): e ects of starvation and subsequent – Food safety using NMR-based metabolomics: assessment of the Atlantic bluefin tuna, feeding on growth and body composition. J. Appl. Ichthyol. 27, 749 754. Thunnus thynnus, from the Mediterranean Sea. Food Chem. Toxicol. 115, 391–397. Liu, Y., Li, Z., Zhang, T., Yuan, J., Mou, Z., Liu, J., 2015a. Growth and energy budget of Carver, J.D., Walker, W.A., 1995. The role of nucleotides in human nutrition. Nutri. juvenile lenok Brachymystax lenok in relation to ration level. Chin. J. Oceanol. – Biochem. 6, 58–72. Limnol. 33 (2), 347 355. Chatzifotis, S., Polemitou, I., Divanach, P., Antonopoulou, E., 2008. Effect of dietary Liu, Y., Ma, D., Xiao, Z., Xu, S., Wang, Y., Wang, Y., Xiao, Y., Song, Z., Teng, Z., Liu, Q., Li, ff taurine supplementation on growth performance and bile salt activated lipase activity J., 2015b. Histological change and heat shock protein 70 expression in di erent fl of common dentex, Dentex dentex, fed a fish meal/soy protein concentrate based diet. tissues of Japanese ounder Paralichthys olivaceus in response to elevated tempera- – Aquaculture 275, 201–208. ture. Chin. J. Oceanol. Limnol. 33, 11 19. ff Christopher, K.B., 2018. Nutritional metabolomics in critical illness. Curr. Opin. Clin. Liu, J., Mai, K., Xu, W., Zhang, Y., Zhou, H., Ai, Q., 2015c. E ects of dietary glutamine on Nutr. Metab. Care 21, 121–125. survival, growth performance, activities of digestive enzyme, antioxidant status and Cloarec, O., Dumas, M.E., Trygg, J., Craig, A., Barton, R.H., Lindon, J.C., Nicholson, J.K., hypoxia stress resistance of half-smooth tongue sole (Cynoglossus semilaevis Günther) – Holmes, E., 2005. Evaluation of the orthogonal projection on latent structure model post larvae. Aquaculture 446, 48 56. limitations caused by chemical shift variability and improved visualization of bio- Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real- – marker changes in 1H NMR spectroscopic metabonomic studies. Anal. Chem. 77 (2), time quantitative PCR and the 2-[Delta][Delta] CT method. Methods 25, 402 408. 517–526. Logan, C.A., Buckley, B.A., 2015. Transcriptomic responses to environmental temperature fi – Coen, M., Lenz, E.M., Nicholson, J.K., Wilson, I.D., Pognan, F., Lindon, J.C., 2003. An in eurythermal and stenothermal shes. J. Exp. Biol. 218, 1915 1924. integrated metabonomic investigation of acetaminophen toxicity in the mouse using Lund, S.G., Lund, M.E.A., Tufts, B.L., 2003. Red blood cell Hsp 70 mRNA and protein as NMR spectroscopy. Chem. Res. Toxicol. 16, 295–303. bio-indicators of temperature stress in the brook trout (Salvelinus fontinalis). Can. J. – Costas, B., Conceição, L.E.C., Aragão, C., Martos, J.A., Ruiz-Jarabo, I., Mancera, J.M., Fish. Aquat. Sci. 60 (4), 460 470.

318 Y. Liu et al. Comparative Biochemistry and Physiology - Part D 29 (2019) 308–319

Mahanty, A., Purohit, G.K., Banerjee, S., Karunakaran, D., Mohanty, S., Mohanty, B.P., Tian, X., Fang, J., Dong, S., 2010. Effects of starvation and recovery on the growth, 2016. Proteomic changes in the liver of Channa striatus in response to high tem- metabolism and energy budget of juvenile tongue sole (Cynoglossus semilaevis). perature stress. Electrophoresis 37, 1704–1717. Aquaculture 310, 122–129. Marine, K.R., Cech Jr., J.J., 2004. Effects of high water temperature on growth, smolti- Trygg, J., Wold, S., 2002. Orthogonal projections to latent structures (O-PLS). J. Chemom. fication, and predator avoidance in juvenile Sacramento River Chinook Salmon. N. 16, 119–128. Am. J. Fish Manag. 24 (1), 198–210. Tsui, T.K.N., Randall, D.J., Chew, S.F., Jin, Y., Wilson, J.M., Ip, Y.K., 2002. Accumulation

McCullough, D.A., Bartholow, J.M., Jager, H.I., Beschta, R.L., Cheslak, E.F., Deas, M.L., of ammonia in the body and NH3 volatilization from alkaline regions of the body Ebersole, J.L., Foott, J.S., Johnson, S.L., Marine, K.R., Mesa, M.G., Petersen, J.H., surface during ammonia loading and exposure to air in the weather loach Misgurnus Souchon, Y., Tiffan, K.F., Wurtsbaugh, W.A., 2009. Research in thermal biology: anguillicaudatus. J. Exp. Biol. 205, 651–659. burning questions for coldwater stream fishes. Rev. Fish. Sci. 17 (1), 90–115. Vandenberg, R.A., Hoefsloot, H.C., Westerhuis, J., Smilde, J.A., Vanderwerf, A.K., 2006. Melis, R., Sanna, R., Braca, A., Bonaglini, E., Cappuccinelli, R., Slawski, H., Roggio, T., Centering, scaling, and transformations: improving the biological information con- Uzzau, S., Anedda, R., 2017. Molecular details on gilthead sea bream (Sparus aurata) tent of metabolomics data. BMC Genomics 7, 142–156. sensitivity to low water temperatures from 1H NMR metabolomics. Comp. Biochem. Viant, M.R., Werner, I., Rosenblum, E.S., Gantner, A.S., Tjeerdema, R.S., Johnson, M.L., Physiol. A 204, 129–136. 2003. Correlation between heat-shock protein induction and reduced metabolic Mou, Z., Li, Y., Xu, G., Zhang, Y., Liu, Y., 2013. A technique of artificial reproduction and condition in juvenile steelhead trout (Oncorhynchus mykiss) chronically exposed to culture in manchurian trout Brachymystax lenok. Chin. J. Fish. 26 (1), 15–18. elevated temperature. Fish Physiol. Biochem. 29, 159–171. Nicholson, J.K., Foxall, P.J., Spraul, M., Farrant, R.D., Lindon, J.C., 1995. 750 MHz 1H Waagbø, R., Jørgensen, S.M., Timmerhaus, G., Breck, O., Olsvik, P.A., 2017. Short-term and 1H–13C NMR spectroscopy of human blood plasma. Anal. Chem. 67, 793–811. starvation at low temperature prior to harvest does not impact the health and acute Olsvik, P.A., Vikeså, V., Lie, K.K., Hevrøy, E.M., 2013. Transcriptional responses to stress response of adult Atlantic salmon. PeerJ 5, e3273. temperature and low oxygen stress in Atlantic salmon studied with next-generation Wang, Y., Liu, Z., Li, Z., Shi, H., Kang, Y., Wang, J., Huang, J., Jiang, L., 2016. Effects of sequencing technology. BMC Genomics 14, 817. heat stress on respiratory burst, oxidative damage and SERPINH1 (HSP47) mRNA Pinotti, L., Baldi, A., Dell'Orto, V., 2002. Comparative mammalian choline metabolism expression in rainbow trout Oncorhynchus mykiss. Fish Physiol. Biochem. 42, with emphasis on the high-yielding dairy cow. Nutr. Res. Rev. 15, 315–331. 701–710. Salze, G.P., Davis, D.A., 2015. Taurine: a critical nutrient for future fish feeds. Yamamoto, T., Koyama, H., Kurajoh, M., Shoji, T., Tsutsumi, Z., Moriwaki, Y., 2011. Aquaculture 437, 215–229. Biochemistry of uridine in plasma. Clin. Chim. Acta 412, 1712–1724. Sanders, B.M., 1990. Stress proteins: potential as multitiered biomarkers. In: Shugart, L., Yap, I.K., Clayton, T.A., Tang, H., Everett, J.R., Hanton, G., Provost, J.P., Le Net, J.L., McCarthy, J. (Eds.), Biomarkers of Environmental Contamination. Lewis Publishers, Charuel, C., Lindon, J.C., Nicholson, J.K., 2006. An integrated metabonomic ap- Boca Raton, Fla, pp. 165–191. proach to describe temporal metabolic disregulation induced in the rat by the model Sanders, B.M., 1993. Stress proteins in aquatic organisms-an environmental perspective. hepatotoxin allyl format. J. Proteome Res. 5, 2675–2684. Crit. Rev. Toxicol. 23, 49–75. Zhou, M., Wang, A., Xian, J., 2011. Variation of free amino acid and carbohydrate con- Silvestre, F., Linares-Casenave, J., Doroshov, S.I., Kültz, D., 2010. A proteomic analysis of centrations in white shrimp, Litopenaeus vannamei:effects of continuous cold stress. green and white sturgeon larvae exposed to heat stress and selenium. Sci. Total Aquaculture 317, 182–186. Environ. 408, 3176–3188.

319